Vascular Endothelial Growth Factor Protein Expression Research Articles

TGF-β2/Smad3 Signaling Pathway Activation Through Enhancing VEGF and CD34 Ameliorates Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats.

Evidence has shown the therapeutic potential of isoflurane (ISO) in cerebral stroke. The present study investigated the mechanism of ISO on vascular endothelial growth factor (VEGF) and CD34 expression in a rat model of stroke. Transient focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) for 1h followed by reperfusion for 24h in rats. ISO was administered for 1.5h when the reperfusion was initiated. Neurologic deficit scores, infarct volumes, HE staining, Nissl staining, and TUNEL staining were evaluated at 24h after reperfusion. The levels of transforming growth factor (TGF)-β2, Smad3, p-Smad3, VEGF, and CD34 proteins were detected by immunofluorescence (IF) staining and Western blot assay. Administration of ISO significantly reduced the neurologic deficit scores, infarct volumes, and damaged and apoptotic cells after cerebral ischemia/reperfusion (I/R) injury (P < 0.05). Meanwhile, ISO post-conditioning significantly increased the expression levels of TGF-β2, p-Smad3, VEGF, and CD34 (P < 0.05), whereas the expression of Smad3 showed no difference (P > 0.05). However, Pirfenidone, a TGF-β2 inhibitor, decreased the expression levels of TGF-β2, p-Smad3, VEGF, and CD34 (P < 0.05). Moreover, the protective effects of ISO post-conditioning were negated by the inhibitor. The present study indicated that ISO attenuates brain damage by activating the TGF-β2/Smad3 signaling pathway and increasing the protein expression of VEGF and CD34 in the rat MCAO model.

The distribution and expression of vascular endothelial growth factor A (VEGFA) during follicular development and atresia in the pig.

The involvement of vascular endothelial growth factor A (VEGFA) in ovarian physiological processes has been widely reported, but the location and role of VEGFA during follicular atresia remain unknown. This study investigated the distribution and expression of VEGFA during porcine follicular development and atresia. Pig ovaries were obtained, individual medium-sized (3-5mm in diameter) antral follicles were separated and classified into healthy, early atretic or progressively atretic groups. Immunobiology and quantitative techniques were used to investigate the varied follicular distribution of VEGFA at both the morphological and molecular level. The results indicated that VEGFA protein expression peaked in tertiary follicles, mostly distributed in the thecal and inner granulosa layers, during follicular development while VEGFA mRNA was mainly expressed in the inner granulosa layers. Additionally, healthy antral follicles showed a significantly higher expression of VEGFA than atretic follicles in both theca and granulosa cells. Knockdown of VEGFA using siRNA revealed an antiapoptosis effect of VEGFA in cultured pig granulosa cells. Our results increase the knowledge of VEGFA functions in follicles.

MiR-223-3p targeting epithelial cell transforming sequence 2 oncogene inhibits the activity, apoptosis, invasion and migration of MDA-MB-468 breast cancer cells.

PurposeThis research was to investigate the role of miR-223-3p targeting epithelial cell transforming sequence 2 oncogene (ECT2) in activity, apoptosis, invasion and migration of MDA-MB-468 breast cancer (BC) cells.MethodsThe human BC cell lines MDA-MB-468 were used for the experiment. They were divided into six groups: blank group (no plasmid transfection), NC group (negative control, transfected empty plasmid), miR-223-3p mimic group (transfected miR-223-3p mimic plasmid), miR-223-3p inhibitor group (transfected miR-223-3p inhibitor plasmid), si-ECT2 group (transfected si ECT2 plasmid) and miR-223-3p mimic+oe-ECT2 group (transfected with miR-223-3p mimic plasmid and ECT2 plasmid).ResultsCompared with the NC group, the mRNA and protein expression of Bax in miR-223-3p mimic and si-ECT2 groups were significantly increased, while the mRNA and protein expression of ECT2, Bcl-2, vascular endothelial growth factor (VEGF), and TGF-β1 were significantly decreased (all P<0.05). Compared with the NC group, the expression of miR-223-3p and the mRNA and protein expression of Bax were significantly decreased in the miR-223-3p inhibitor group, while the mRNA and protein expression of ECT2, Bcl-2, VEGF and TGF-β1 were significantly increased (both P<0.05). Compared with the single processing group, the mRNA and protein expression of Bax in the miR-223-3p mimic+si-ECT2 group were significantly increased, while the mRNA and protein expression of ECT2, Bcl-2, VEGF, and TGF-β1 were significantly decreased (all P<0.05).ConclusionMiR-223-3p targets and inhibits the expression of ECT2, thus inhibiting the invasion and migration of BC cells, and promoting cell apoptosis. miR-223-3p plays a protective role in BC.

Notch Signaling Pathway Is Involved in bFGF-Induced Corneal Lymphangiogenesis and Hemangiogenesis.

Background Notch/Dll4 involvement in cornea neovascularization (CRNV) and lymphangiogenesis is unclear. This study aimed to explore the role of notch signaling in basic fibroblast growth factor- (bFGF-) induced corneal lymphangiogenesis and hemangiogenesis. Methods Corneal stroma of C57BL/6 mice was implanted with bFGF- or phosphate-buffered saline- (PBS-) soaked pellets. Corneal lymphangiogenesis and neovascularization were evaluated by immunofluorescence. Vascular endothelial growth factor-A (VEGF-A), Delta-like ligand 4 (Dll4), and Notch1 mRNA and protein expression were examined on days 1, 3, 7, and 14 by real-time polymerase chain reaction and western blot. Corneal cells were treated with ranibizumab, dexamethasone, and γ-secretase inhibitor (GSI). Microspheres were used to evaluate corneal hemangiogenesis in vivo. Results Corneal hemangiogenesis reached its peak on day 7 after bFGF implantation, and corneal lymphangiogenesis was significantly higher on day 7 and 14, compared with PBS. mRNA and protein expression of VEGF-A, Dll4, and Notch1 were higher in bFGF-induced animal models compared with controls. Corneal hemangiogenesis and lymphangiogenesis decreased after 7 days of ranibizumab or dexamethasone treatment. After adding GSI for 24 h in bFGF-induced cells, the expression of Notch1 and Dll4 were downregulated compared with that in the control group whereas the expression level of VEGF-A was upregulated. Fluorescent particle number was higher in the GSI group. Ranibizumab and dexamethasone decreased the fluorescence signal. Conclusion The notch signaling pathway plays a role in regulating VEGF expression, affecting corneal lymphangiogenesis and hemangiogenesis in mice. The molecular imaging probe technique can visualize the changes in the VEGF-A expression level of corneal limbus hemangiogenesis.

Expression and significance of MiR-126 and VEGF in proliferative diabetic retinopathy.

To study the expression and significance of micro ribonucleic acid (miR)-126 and vascular endothelial growth factor (VEGF) in the vitreous body and proliferative membrane tissues of affected eyes and plasma in proliferative diabetic retinopathy (PDR). A total of 50 PDR patients admitted to our hospital from January 2017 to January 2018 were collected, including 17 cases in stage IV, 20 cases in stage V, and 13 cases in stage VI according to the DR staging criteria. Another 30 patients with an idiopathic macular hole were selected as control group. After admission, the examinations were performed, venous blood was drawn, and vitrectomy was performed, during which the vitreous body and proliferative membrane tissues were taken. The plasma VEGF content was detected via enzyme-linked immunosorbent assay (ELISA). The VEGF protein expression level was detected via Western blotting in vitreous body and proliferative membrane tissues. Moreover, the miR-126 expression was detected via quantitative polymerase chain reaction (qPCR). In the vitreous body tissues, proliferative membrane tissues and plasma, the expression level of miR-126 was significantly lower in control group than that in stage IV, V, and VI groups (p<0.05). The miR-126 expression was significantly lower in stage IV group than that in stage V and VI groups (p<0.05), meanwhile, miR-126 expression was significantly lower in stage V group than that in stage VI group (p<0.05). In the vitreous body tissues, proliferative membrane tissues and plasma, the VEGF mRNA and protein expression levels were significantly higher in control group than those in stage IV, V, and VI groups (p<0.05). The VEGF expressions were significantly higher in stage IV group than those in stage V and VI groups (p<0.05). In addition, VEGF expressions were significantly higher in stage V group than those in stage VI group (p<0.05). Furthermore, the VEGF expression level was negatively correlated with the miR-126 expression level. We found that the abnormally high expression of miR-126 negatively regulates VEGF, which may be one of the important mechanisms of occurrence of PDR. The miR-126 and VEGF content can serve as important predictors for the condition of PDR.

Liraglutide Increases VEGF Expression via CNPY2-PERK Pathway Induced by Hypoxia/Reoxygenation Injury.

Liraglutide (Lir) is a glucagon-like peptide-1 receptor agonist that lowers blood sugar and reduces myocardial infarct size by improving endothelial cell function. However, its mechanism has not yet been clarified. Unfolded protein response (UPR) plays an important role in the pathogenesis of myocardial ischemia-reperfusion injury. It determines the survival of cells. Endoplasmic reticulum position protein homologue 2 (CNPY2) is a novel initiator of UPR that also participates in angiogenesis. To this extent, the current study further explored whether Lir regulates angiogenesis through CNPY2. In our article, a hypoxia/reoxygenation (H/R) injury model of human umbilical vein endothelial cells (HUVECs) was established and the effect of Lir on HUVECs was first evaluated by the Cell Counting Kit-8. Endothelial tube formation was used to analyze the ability of Lir to induce angiogenesis. Subsequently, the effect of Lir on the concentrations of hypoxia-inducible factor 1α (HIF1α), vascular endothelial growth factor (VEGF), and CNPY2 was detected by enzyme-linked immunosorbent assay. To assess whether Lir regulates angiogenesis through the CNPY2-initiated UPR pathway, the expression of UPR-related pathway proteins (CNPY2, GRP78, PERK, and ATF4) and angiogenic proteins (HIF1α and VEGF) was detected by reverse transcription-polymerase chain reaction and Western blot. The results confirmed that Lir significantly increased the expression of HIF1α and VEGF as well as the expression of CNPY2-PERK pathway proteins in HUVECs after H/R injury. To further validate the experimental results, we introduced the PERK inhibitor GSK2606414. GSK2606414 was able to significantly decrease both the mRNA and protein expression of ATF4, HIF1α, and VEGF in vascular endothelial cells after H/R injury. The effect of Lir was also inhibited using GSK2606414. Therefore, our study suggested that the CNPY2-PERK pathway was involved in the mechanism of VEGF expression after H/R injury in HUVECs. Lir increased the expression of VEGF through the CNPY2-PERK pathway, which may promote endothelial cell angiogenesis and protect HUVEC from H/R damage.

Effects of Carvacrol in an Experimentally Induced Esophageal Burn Model: Expression of VEGF and Caspase-3 Proteins

Introduction We investigated the therapeutic effects of carvacrol in an experimental esophageal burn rat model with immunohistochemical techniques. Materials and Methods: Three groups were included in this study, composed of eight Wistar albino rats each. The control group was given 1 mL 0.9% (wt/vol) NaCl; esophageal burns were induced in groups 2 and 3 by administration of 1 mL 40% NaOH in the distal 2 cm of the esophagus. The treatment group was administered 75 mg/kg carvacrol in 2 mL 0.9% NaCl for 10 days. After a routine histological examination of the tissues, sections were stained with vascular endothelial growth factor (VEGF) and caspase-3 for immunohistochemical analysis and were examined under a light microscope. Results: In the control group, there were regular cells in the cornified epithelial tissue and cylindrical cells in the basal layer, which faced toward the apical surface in the mitotic phase. The burn group displayed wide degeneration, necrosis, and abundant apoptotic cells in the epithelial tissue as well as intense inflammatory cell infiltration. In the treatment group, there was an increase in mitotic activity in the basal cells of the epithelial layer and degenerative changes, but a preserved epithelial layer and significant cornified structures. The treatment group showed positive caspase-3 expression in some apoptotic cells within the epithelial layer and in connective tissue, and there were only a small number of degenerated cells in the muscle layer. Additionally, in the treatment group, VEGF expression was evident in small numbers of inflammatory cells in the papillary region of the epithelium, and in dilated vascular endothelial cells. Conclusions: Carvacrol may contribute to a reduction in fibrosis by decreasing inflammation and preventing cell apoptosis.

Hypertonic saline downregulates endothelial cell-derived VEGF expression and reduces blood-brain barrier permeability induced by cerebral ischaemia via the VEGFR2/eNOS pathway

The aim of the present study was to explore the possible mechanisms by which hypertonic saline (HS) effectively ameliorates cerebral oedema via the vascular endothelial growth factor receptor 2 (VEGFR2)-mediated endothelial nitric oxide synthase (eNOS) pathway of endothelial cells in rats. A middle cerebral artery occlusion (MCAO) model in Sprague-Dawley rats and an oxygen-glucose deprivation (OGD) model in cells were used in the present study. Evans blue (EB) staining and a horseradish peroxidase flux assay were performed to evaluate the protective effect of 10% HS on the blood-brain barrier (BBB). The expression levels of vascular endothelial growth factor (VEGF), VEGFR2, zonula occludens 1 (ZO1) and occludin were quantified. The results demonstrated that 10% HS effectively reduced EB extravasation in the peri-ischaemic brain tissue. At 24 h after MCAO, the protein expression levels of VEGF and VEGFR2 in the peri-ischaemic brain tissue were downregulated following treatment with 10% HS. In vitro experiments demonstrated that the permeability of a monolayer endothelial cell barrier was decreased significantly following HS treatment. In addition, VEGF and VEGFR2 protein expression levels were increased in endothelial cells under hypoxic conditions, but that effect was suppressed by HS treatment. Furthermore, HS inhibited the downregulation of ZO1 and occludin effectively, possibly through the VEGFR2/phospholipase C γ1 (PLCγ1)/eNOS signalling pathway. In conclusion, 10% HS may alleviate cerebral oedema through reducing ischaemia-induced BBB permeability, as a consequence of inhibiting VEGFR2/PLCγ1/eNOS-mediated downregulation of ZO1 and occludin.

Correlation between endometrial receptivity with expressions of IL-1 and VEGF in rats with polycystic ovary syndrome.

To investigate the correlation between endometrial receptivity with expressions of interleukin-1 (IL-1) and vascular endothelial growth factor (VEGF) in rats with polycystic ovary syndrome (PCOS). A total of 24 female Sprague-Dawley (SD) 21 days old were randomly divided into control group (n=12) and PCOS group (n=12). Rats in the control group were normally raised, and PCOS model was established in rats of the PCOS group. All the rats were sacrificed when they grew to 80 days old. Immunohistochemistry was applied to detect the expressions of IL-1 and VEGF. Western blotting was performed to measure the relative expressions of IL-1 and VEGF proteins. Quantitative Polymerase Chain Reaction (qPCR) was utilized to determine the relative messenger ribonucleic acid (mRNA) expressions of IL-1 and VEGF. Data related to endometrial receptivity were detected. The expression levels of IL-1 and VEGF in the PCOS group declined markedly compared with those in the control group, and the differences were statistically significant (p<0.05). PCOS group had notably lower protein expressions of IL-1 and VEGF than the control group, with statistically significant differences (p<0.05). The mRNA expressions of IL-1 and VEGF in PCOS group were significantly lower than those in the control group, displaying statistically significant differences (p<0.05). Compared with that in the control group, the endometrial receptivity of rats in the PCOS group was reduced evidently, and the difference was statistically significant (p<0.05). Both IL-1 and VEGF had positive correlations with the endometrial receptivity. Both IL-1 and VEGF are positively correlated with the endometrial receptivity in the case of PCOS, which can serve as therapeutic targets for PCOS and improve endometrial receptivity in the future.

Abstract LB-131: Mast cells promote local angiogenic and structural remodeling, a precursor to prostate cancer

Abstract Prostate cancer (PCa) is the second cancer-cause of death of men in the USA. Upon transformation, prostatic epithelial cells (EC) gradually evolve from benign, premalignant, to malignant phenotypes. Prostatic Intraepithelial Neoplasia (PIN) is an abnormal growth of EC progressing from Low grade (L) PIN to High grade (H) PIN. Twenty percent of patients with HPIN advance to PCa, therefore, most studies focus on the HPIN-PCa progression. We hypothesized that the less-studied LPIN stage may feature unique remodeling processes that also predispose to PCa. Perivascular and prostate-resident mast cells (MC) are first-line sources of preformed tumor-promoting mediators, including angiogenic vascular endothelial growth factor-A (VEGF) and sphingosine-1-phosphate (S1P), a sphingolipid metabolite produced by sphingosine kinase (SphK). Using the C3(1)/SV40Tag transgenic (C3) mouse model that mimics human PCa initiation and progression, we sought to investigate remodeling in LPIN. We designed a novel quantitative method of image analysis to measure in situ angiogenesis in prostate sections that integrates capillary-restricted CD31 quantification and morphometric parameters. Increased angiogenesis was discovered in LPIN and MC were located near the newly generated blood capillaries. LPIN sections featured augmented MC numbers, more activated in LPIN, than normal C3 or wild-type littermate sections. Moreover, local mRNA coding for Sphk, Vegfa and matrix metalloproteinase (Mmp) 9 were significantly increased in LPIN samples, accompanied with elevation of S1P. Additional evidence of structural remodeling in LPIN samples included a substantial increase in mRNA coding for collagens type I and III (Col1a1 and Col3a1). Importantly, we established a novel computer-aided imaging method to quantify cell-associated VEGF protein expression in tissue sections based on signal intensity measured through conversion to a gray-scale value then area-under-the-curve calculation. Remarkably, local VEGF expression was exclusively restricted to MC in all experimental groups and was more elevated in LPIN samples. Next, transcriptomics for remodeling genes conducted with human patient samples revealed that, for each patient, cancer-bearing biopsies also featured significant increases in SPHK, VEGF, MMP9, COL1 and COL3 mRNA expressions, compared to normal biopsies. Altogether, our preclinical and human data provides a new strategic paradigm to untangle the link between MC, early local remodeling observed in LPIN and PCa. Surveillance of MC and their mediators may lead to new diagnostic and precision medicine modalities for the clinical targeting of MC and for early interception of PCa. Citation Format: Ahmed Aladhami, Michael Hobensack, Nabihah I. Kumte, Alena P. Chumanevich, Taryn L. Cranford, Alexander Sougiannis, Reilly T. Enos, Kandy T. Velázquez, Ioulia Chatzistamou, John W. Fuseler, Angela Murphy, Carole A. Oskeritzian. Mast cells promote local angiogenic and structural remodeling, a precursor to prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-131.

Evaluation of Angiogenesis Process after Metformin and LY294002 Treatment in Mammary Tumor.

The angiogenesis process is regulated by many factors, such as Hypoxia-Inducible Factor-1 (HIF-1) and Vascular Endothelial Growth Factor (VEGF). Metformin has demonstrated its ability to inhibit cell growth and the LY294002 is the major inhibitor of PI3K/AKT/mTOR pathway that has antiangiogenic properties. Canine mammary tumor cell lines CMT-U229 and CF41 were treated with metformin and LY294002. Cell viability, protein and gene expression of VEGF and HIF-1 were determined in vitro. For the in vivo study, CF41 cells were inoculated in female athymic nude mice treated with either metformin or LY294002. The microvessel density by immunohistochemistry for CD31 as well as the gene and protein expression of HIF-1 and VEGF were evaluated. The treatment with metformin and LY294002 was able to reduce the cellular viability after 24 hours. The protein and gene expression of HIF-1 and VEGF decreased after treatment with metformin and LY294002. In the in vivo study, there was a decrease in tumor size, protein and gene expression of HIF-1 and VEGFA, in addition to the decreasing of CD31 expression after all treatments. Our results demonstrate the effectiveness of metformin and LY294002 in controlling the angiogenesis process in mammary tumors by VEGF and HIF-1, the most important angiogenic markers.

Isoflurane-Induced Postoperative Neurovascular and Cognitive Dysfunction Is Associated with VEGF Overexpression in Aged Rats.

Postoperative cognitive dysfunction (POCD) is a common complication in older adults; however, its aetiology remains unclear. Although vascular endothelial growth factor (VEGF) is associated with blood-brain barrier (BBB) disorders and neurological disease, its role in POCD is unknown. Here, we investigated the effect of brain VEGF inhibition on isoflurane-induced cognitive impairment in an aged rat model of POCD. VEGF protein expression was increased in the hippocampus after isoflurane exposure, suggesting that inhalation anaesthesia induces hippocampal VEGF protein overexpression in aged rats. Pretreatment with 2mg/kg RB-222, an anti-VEGF neutralizing antibody, may partially abolish the degradation of occludin protein in cerebral capillaries, thereby maintaining the ultrastructural and functional integrity of the hippocampal BBB. Inhibition of VEGF also significantly attenuated the isoflurane-induced cognitive deficits in the Morris water maze task. Together, our findings show, for the first time, that elevated expression of brain VEGF after isoflurane exposure contributes to POCD in aged rats. Therefore, therapeutic strategies involving VEGF should take into consideration its role in the pathogenesis of POCD.

MiRNAs Are Involved in Tall Cell Morphology in Papillary Thyroid Carcinoma.

Five percent of papillary thyroid carcinomas (PTC) show an adverse clinical outcome (ACO). The tall cell variant of papillary thyroid carcinomas (TCV) is a good predictor of an ACO, however, the identification of tall-cells is subjective. Micro RNAs are short non-coding ribonucleic acids (miRNA). Their expression in PTC could be a powerful, more objective predictor of prognosis. Methods: Forty-four PTC underwent miRNA profiling, twenty-four of them were TCV. The miRNA dataset was validated by analysis of expression of known target proteins (vascular endothelial growth factor (VEGF) and phosphatase and tensin homolog (PTEN)) in 125 patients including 48 TCV and 57 with an ACO. Results: One hundred and forty-nine miRNAs were significantly associated with an ACO, seventy-one of them with TC-morphology. Twenty-two miRNAs were identified as targets for VEGF and thirty-two as targets for PTEN. In univariate and multivariable analysis, reduced expression of PTEN and an increased expression of VEGF were associated with shorter relapse free survival. A classifier, including TC-morphology, pT-stage, VEGF, and PTEN, predicted relapse with an 80% accuracy. Conclusions: Some miRNAs predict outcome in PTC and are involved in TC-morphology in PTC. These miRNAs may serve as more objective indicators of an ACO than tall cell morphology. PTEN and VEGF protein expression are prognostically relevant and are at least partially regulated by miRNAs.

Tumor necrosis factor-related apoptosis-inducing ligand modulates angiogenesis and apoptosis to inhibit non-small cell lung carcinoma tumor growth in mice.

ObjectiveTo investigate the anti-tumor effect and mechanism of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in non-small cell lung carcinoma (NSCLC) in mice.MethodsWe first established NSCLC animal models using 20 BALB/c nude mice that were randomly divided into two equal groups (n = 10): TRAIL-treated and control untreated groups. We measured expression levels of B cell leukemia/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), vascular endothelial growth factor (VEGF), and VEGF receptor (VEGFR). We also performed microvessel density, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunohistochemical assays to determine the effect of TRAIL on apoptosis and angiogenesis in NSCLC tumors in vitro.ResultsTRAIL inhibited tumor growth in the NSCLC mouse model, and the TUNEL assay showed that it induced tumor cell apoptosis. Immunohistochemical staining revealed that TRAIL induced Bcl-2 protein downregulation, suggesting that the mitochondrial apoptotic pathway is involved in regulating NSCLC apoptosis. However, TRAIL did not affect Bax protein expression. Immunohistochemical staining also revealed significantly reduced VEGF and VEGFR protein expression in the TRAIL group, indicating that TRAIL limits angiogenesis in NSCLC tumor tissues.ConclusionsIn conclusion, TRAIL inhibits NSCLC growth both by inducing tumor cell apoptosis and restricting angiogenesis in tumors.

Ultrasound-mediated destruction of vascular endothelial growth factor (VEGF) targeted and paclitaxel loaded microbubbles for inhibition of human breast cancer cell MCF-7 proliferation

AimsVascular endothelial growth factor (VEGF) can promote cell division, proliferation and migration. In this study, we aimed to investigate roles of ultrasound-mediated destruction of VEGF-targeted and paclitaxel (PTX)-loaded lipid microbubbles (VTPLLM + US) in human breast cancer cells. MethodsThe activity of MCF-7 cells was determined by cell counting Kit-8. Flow cytometry was performed to detect the cells apoptosis and cell cycle. The expression of cell cycle-associated proteins, matrix metalloprotein-9 (MMP-9), VEGF and apoptosis-associated proteins were detected by qRT-PCR and Western blot. ResultsThe obtained data suggested that VTPLLM + US promoted the G1 phase cell cycle arrest and suppressed the viability of MCF-7 cells. We also found that VTPLLM + US accelerated cells apoptosis. Cell cycle-associated proteins and VEGF expression were modulated by VTPLLM + US. Moreover, VTPLLM + US was found to regulate the expression levels of apoptosis-associated proteins in MCF-7 cells. Our findings suggested that VTPLLM + US suppressed the proliferation and accelerated the apoptosis of MCF-7 cells through regulating VEGF expression. ConclusionsThe potential effects of VTPLLM + US on apoptosis of MCF-7 cells suggest that applying VTPLLM + US might be an effective strategy in breast cancer therapies.

Administration of human umbilical cord blood mononuclear cells restores bladder dysfunction in streptozotocin-induced diabetic rats.

This study evaluated the effect of human umbilical cord blood mononuclear cells (HUCB-MNCs) on bladder dysfunction in streptozotocin (STZ; 35 mg/kg, i.v.)-induced diabetic rats. Adult male Sprague-Dawley rats (n = 30) were equally divided into three groups: control group, STZ-diabetic group, and HUCB-MNC-treated group (1 × 106 cells). HUCB-MNCs were isolated by density gradient centrifugation from eight healthy donors and injected into the corpus cavenosum in STZ-diabetic rats 4 weeks after the induction of diabetes. Studies were performed 4 weeks after HUCB-MNC or vehicle injection. In vitro organ bath studies were performed on bladder strips, whereas protein expression of hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), and α-smooth muscle actin (SMA) in the bladder and the ratio of smooth muscle cells (SMCs) to collagen were determined using western blotting and Masson trichrome staining. Neurogenic contractions of detrusor smooth muscle strips were 55% smaller in the diabetic group than control group (P < 0.05); these contractions were normalized by HUCB-MNC treatment. In addition, HUCB-MNC treatment restored the impaired maximal carbachol-induced contractile response in detrusor strips in the diabetic group (29%; P < 0.05). HUCB-MNC treatment improved the KCl-induced contractile response in the diabetic bladder (68%; P < 0.05), but had no effect on ATP-induced contractile responses. Increased expression of HIF-1α and VEGF protein and decreased expression of α-SMA protein and the SMC/collagen ratio in diabetic rats were reversed by HUCB-MNC. Administration of HUCB-MNCs facilitates bladder function recovery, which is likely related to downregulation of HIF-1α expression and attenuation of fibrosis in STZ-diabetic rats.

Inhibitory Effect of Semen Litchi Drug Serum on the Proliferation of Human Hepatoma HepG2 Cells and Expression of VEGF and MMP-9.

To investigate inhibitory effect of semen litchi drug serum on proliferation of human hepatoma HepG2 cells and its effect on the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9). An experimental study. College of Pharmacy, Guangxi University of Chinese Medicine, China, from June 2017 to January 2018. Semen litchi drug serum with concentrations of 0 mg/kg (control group), 3 g/kg (low dose group), 6 g/kg (medium dose group) and 12 g/kg (high dose group) was used to act on HepG2 cells at the logarithmic phase. Inhibitory effect of semen litchi drug serum on cell growth, expression of VEGF and MMP-9 mRNA and protein was detected. Inhibitory effect of semen litchi drug serum on the proliferation of HepG2 cells significantly increased with the increase of drug concentration, which was dose-time dependent. Expression levels of VEGF and MMP-9 mRNA in HepG2 cells after 48 hours of treatment by semen litchi low-dose group, medium-dose group, and high-dose group were lower than those in control group (all p <0.001). After acting on HepG2 cells for 48 hours, relative expressions of VEGF and MMP-9 protein in semen litchi low-dose group, medium-dose group, and high-dose group were lower than those in control group (all p<0.001). Semen litchi drug serum can inhibit proliferation of hepatoma cells in vitro. The anti-hepatoma effect of semen litchi drug serum may be exerted through down-regulating the expression of VEGF and MMP-9 and inhibiting angiogenesis of hepatocellular carcinoma.

Expression and significance of hypoxia-inducible factor-1α and vascular endothelial growth factor and receptor 2 in stage 3 pressure injury of rats

To analyze the expression and relationship of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR) in local skin tissues of pressure injury and investigate the possible mechanism of stage 3 pressure injury refractory wound. Forty male SD rats were randomly divided into normal control group, compressed 3 d, 5 d, 7 d, and 9 d groups. Stage 3 pressure injury animal model were established by magnet compression. The morphology of skin was observed by HE staining. The expression of VEGF was detected by immunohistochemistry. The expression levels of HIF-1α, VEGF and KDR protein in skin tissue were detected by Western blot. One-way analysis of variance and LSD test were performed on the data. ①The HE results showed that compared with the normal control group, the epidermis of the compressed group was gradually thickened, the number of blood vessels was decreased, the collagen arrangement disordered and inflammatory cells infiltration were increased. ②Immunohistochemical results showed that the expression of VEGF protein in the 3 d group was significantly higher than that in the normal control group (P＜0.01). The expression of VEGF protein in the skin tissue of 5 d, 7 d and 9 d groups was lower than that in normal control group (P＜0.05). WB results were consistent with immunohistochemistry results. ③WB results showed that the expression of HIF-1α in the skin tissues of the rats in 3 d, 5 d and 7 d groups was higher than that in the normal control group (P＜0.01 or P＜0.05). The expression of KDR protein was lower than that of the normal control group (P＜0.05 or P＜0.01). HIF-1α mediated reduction of VEGF and KDR protein expression and decreased tissue angiogenesis may be one of the important causes of chronic dysfunction of stage 3 pressure injury.

The Effect and Mechanism of Vascular Endothelial Growth Fac-tor (VEGF) on Tumor Angiogenesis in Gallbladder Carcinoma

To investigate the effect of vascular endothelial growth factor (VEGF) on tumor angiogenesis in gallbladder carcinoma. Fifty one patients with gallbladder carcinoma were enrolled as observation group. Thirty healthy people were included as control group. Chemically synthesized siRNA sequences targeting VEGF was transfected with VEGF-siRNA. A blank group (group B), a negative control group (transfected with independent sequence, group C), and an inhibition group (transfected with VEGF siRNA, group D) were established. Physiological saline was set as group A. The expression of VEGF was detected by qRT-PCR. The expression of VEGF protein was detected by Western blot. MVD was used to measure microvessel density. CCK-8, Transwell and flow cytometry were used to detect cell proliferation, invasion and apoptosis. The tumor volume of nude mice and VEGF mRNA expression in group D was significantly smaller than that in group B and C (P<0.05). The MVD density in group B and C was significantly higher than that in group D (P<0.01). The proliferation of cells was detected from the 3rd day, and the proliferation of cells in the blank and negative control groups was faster than that of the inhibition group (P<0.05). The apoptosis rate of the blank group and the negative control group was lower than that of the inhibition group (P<0.001). VEGF is highly expressed in serum of patients with cholangiocarcinoma, it promotes angiogenesis, proliferation and invasion of gallbladder cancer cells, and inhibits apoptosis of tumor cells.

Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis.

Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17. Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development.